This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The proteasome is the major cellular protease. It is involved in the controlled degradation of proteins that regulate a wide variety of cellular processes, such as transcription, apoptosis, cell division and DNA repair. With an important role in homeostasis of so many proteins it is not surprising that observed increased proteasome activity (e.g. in multiple myelomas) or decreased proteasome activity (e.g. in many neurodegenerative diseases) is a pathological factor in many diseases. One determinant of cellular proteasome activity is the level of proteasomes in the cell. Thus, it is important to understand how cellular proteasome levels are regulated and how proteasome assembly is regulated. The long-term goal of this project is to understand the mechanisms of proteasome assembly. Recent work has identified four chaperones that facilitate the formation of proteasome regulatory particle (RP). Each chaperone binds to the C-domain of a specific AAA-ATPases located in the RP. Despite this similarity in function and binding properties, there is no sequence or structural conservation among these chaperones. The objective of the research proposed here is to understand the role each RP chaperone plays in the formation of proteasomes. We hypothesize that the chaperones act as templates for specific base subunits in the assembly of RP. Secondly, they regulate the order in which precursor complexes assemble in a spatial and temporal manner. We also hypothesize that the structural difference among the chaperones are important to accommodate as well as prevent a unique set of interactions for specific ATPases during the assembly process. We will use in vitro binding and reconstitution assays to study this. We furthermore will obtain structural information of the chaperones in combination with the C-domain they bind to. We expect that information from the experiments proposed here will provide a detailed understanding of the mechanisms the chaperones employ to assist proteasome assembly. This could potentially provide new drug targets and ultimately enable us to manipulate proteasome levels or activity for therapeutic means.